The present invention relates to a nucleic acid construct for expressing active substances which can be activated by proteases and to its preparation and use.
Like inflamed areas, tumors are distinguished from the surrounding normal tissue by a substantial increase in the formation and secretion of proteases [Schmitt et al., Fibrinol. 6, 3 (1992), Cottam et al., Int. J. Oncol. 2, 861 (1993), Tryggvason et al., Breast Cancer Res. And Treatm. 24, 209 (1993), Leto et al., Anticancer Res. 12, 235 (1992), Hart, Fibrinol. 6, 11 (1992), Albini et al., J. Natl. Cancer Inst. 83, 735 (1991)]. Examples of these proteases are plasminogen activators, cathepsins and matrix metalloproteinases.
An essential function of these tumor proteases is to dissolve the extracellular matrix to allow the tumor cells to invade, and grow in an infiltrative manner in, normal tissue. At the same time, these proteases protect the tumor from the defence mechanisms of the body insofar as the active compounds which are required for defence are cleaved, and thereby inactivated, by the proteases which are formed by the tumor. Thus, for example, antibodies, cytokines and growth factors, complement factors, coagulation factors and mediators are inactivated by tumor proteases.
In the past, the aim was, therefore, to inhibit the infiltrative growth and metastatic growth of tumors, and inactivation of the defence mechanisms of the body, by inhibiting the tumor cell proteases [Hocman, Int. J. Biochem. 24, 1365 (1992), Troll et al., JNCI 73, 1245 (1984), Ray et al., Eur. Respir. 7, 2062 (1994), Koop et al., Cancer Res. 54, 4791 (1994), Chiriri et al., Int. J. Cancer 58, 460 (1994), Denhardt et al., 59, 329 (1993), Melchiori et al., Cancer Res. 52, 2353 (1992)]. However, particularly for stoichiometric and pharmacokinetic reasons, little success has previously been achieved in inhibiting tumor cell proteases.
An attempt was therefore made to use the tumor cell proteases to activate bacterial toxins such as Staphylococcus aureus .alpha.-hemolysin [Panchal et al., Nature Biotechn. 14, 852 (1996)]. For this, an amino acid sequence (SEQ ID NO:1), i.e. XX-Arg-X, was inserted into positions 129 to 132 of the .alpha.-hemolysin and in this way inactive mutants were produced which are only cleaved, and thereby activated, by tumor proteases such as cathepsin B.
Based on these results, proimmunolysins were proposed [Panchal et al., Nature Biotechn. 14, 852 (1996)], which proimmunolysins comprise an antibody which is coupled to a Staphylococcus aureus .alpha.-hemolysin which can be activated by tumor proteases or to a sea anemone equinatoxin II, with the antibody determining the target cell specificity of the coupling product.
However, the proposed concept suffers from the following disadvantages in relation to its use in tumor therapy:
In the first place, the authors chose xenogeneic nonendogenous lysins and/or toxins which are immunogenic for the host organism (for example, patients) and as a result induce an immune reaction in the host organism, which immune reaction neutralizes and inactivates the antibody/toxin conjugate. In the second place, it is known [Sedlacek et al., Antibodies as Carriers of Cytotoxicity, Contrib. to Oncol. 43, Karger Verlag, Munich, 1992] that, due to their molecular size and to the rheological conditions at the tumor, tumor-specific antibodies and immunotoxins only accrue in very small quantities (0.01-0.001% of the given antibody or immunotoxin/g of tumor) at the tumor and only penetrate the tumor to an incomplete extent so that it is either not possible to destroy all the tumor cells or only possible to destroy a small portion of the cells of a tumor. Then again, the extent to which tumor antigens, against which the antibody is directed, are expressed usually differs between the individual tumor cells, and the variable, antigen-negative tumor cells readily evade the attack by the antibodies or the immunotoxins. In addition to this, antigens which are secreted by the tumor cells neutralize the antibodies at the periphery of the tumor (Sedlacek et al., Monoclonal Antibodies in Tumor Therapy, Contrib. to Oncol., Karger Verlag, 1988).
Consequently, there is still a great need for a target cell-specific therapy for tumors and inflammations.